Our long term goal is to establish the structural and mechanistic basis for force production by biological motors. The specific aims of this proposal are to establish the kinetic and thermodynamic basis of force generation of the Eg5 and Kar3 ATPases in direct comparison to Ncd and conventional kinesin. All four are kinesin superfamily molecular motors that use ATP to drive unidirectional microtubule-based movements. Ncd, Eg5, and Kar3 are all involved in spindle dynamics during meiosis and/or mitosis and therefore are required for proper chromosome segregation. In contrast, kinesin is a neuronal motor that drives movements of membranous organelles. Kinesin's motility is distinctive because of its processivity. Ncd, Kar3, and Eg5 are believed not to be processive, and their motility is significantly slower than that promoted by kinesin. Both Ncd and Kar3 promote minus-end directed microtubule movements, yet kinesin and Eg5 promote plus-end directed movements. Furthermore, Kar3 as a monomer exhibits unidirectional movement. Because Kar3 is monomeric and can generate unidirectional movement, Kar3 is an interesting motor to study in direct comparison to the dimeric kinesins-- kinesin, Eg5, Ncd. Our results with Ncd and kinesin indicate that both motor domains of the dimer are required for movement. Eg5 is also dimeric, yet evidence to date indicates it is not processive. As a spindle motor, its mechanism may be more similar to Ncd's even though Ncd motility is directed to the minus-end of microtubules. Our studies with Eg5 in direct comparison to Ncd and kinesin will define the mechanistic features required specifically for processivity that may be distinct from those features that drive plus-end directed movements. The experiments will evaluate the mechanistic features that spindle motors have in common, and at the same time address specific questions about energy transduction for dimeric motors in comparison to monomeric motors. A comprehensive kinetic and thermodynamic analysis of these 4 molecular motors will provide rigorous and direct information to begin to understand the structural and mechanistic requirements for the diverse movements occurring during the cell cycle where genetic alteration can result in birth defects and diseases such as cancer.